Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that causes a diverse array of diseases ranging from recurrent cold sores and genital lesions to blindness and encephalitis. The pathogenesis of HSV-1 depends both on the virus's ability to replicate and its ability to spread. The HSV-1 VP22 protein, encoded by the UL49 gene, has been shown to play an important role in both virus production and spread. Our studies show a UL49-null virus possesses an intriguing and unusual phenotype -- that of normal protein synthesis at early times in infection followed by a synchronous and nearly-global shutdown in protein synthesis at late times in infection. The defect is in protein synthesis and not protein stability. This same virus possesses a defect in the accumulation of a subset of viral mRNAs at early times in infection. The defects in protein synthesis and mRNA accumulation are distinct and separable with regards to both timing during infection and the genes affected. As these findings are novel, nothing is known regarding the mechanisms by which VP22 regulates protein synthesis at late times and mRNA accumulation at early times in infection. The data obtained in the proposed studies will identify those mechanisms and serve as a solid foundation for a future R01 application in which we will molecularly dissect VP22's roles in protein synthesis and mRNA accumulation. Our first aim is to identify the mechanism(s) responsible for the protein synthesis shutdown observed in UL49-null virus infected cells at late times in infection. We will determine whether decreased mRNA export, decreased translation, or both are responsible for the decreased levels of protein synthesis observed late in UL49-null virus infections. We will also identify proteins that undergo decreased synthesis late in UL49-null virus infections. Finally, we will determine whether VP16 and vhs are involved in VP22-dependent protein synthesis. VP22, VP16, and vhs form a complex and secondary mutations that alleviate the UL49-null virus-associated spread defect frequently occur in vhs. Thus, we hypothesize VP22 acts in concert with VP16 and/or vhs to regulate protein synthesis and will test this hypothesis through targeted mutational analyses. Our second aim is to identify the mechanism(s) responsible for the decreased steady-state mRNA levels observed at early times in UL49-null virus infections. We will determine whether increased mRNA degradation, decreased transcription, or both are responsible for the decreased steady-state mRNA levels observed early in UL49-null virus infections. We will perform HSV-1 microarray studies to identify mRNAs present in decreased levels early in UL49-null virus infections. Finally, we hypothesize that VP22 acts to increase mRNA accumulation early in infection by modulating the activity of vhs, either independently or in concert with VP16. We will test this hypothesis through targeted mutational analyses. PUBLIC HEALTH RELEVANCE: Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that causes a diverse array of diseases ranging from recurrent cold sores and genital lesions to blindness and encephalitis. The pathogenesis of HSV-1 depends both on the virus's ability to replicate itself and its ability to spread. The studies proposed herein will enable us to elucidate mechanisms utilized by HSV-1 for virus replication and spread as a prerequisite to the development of improved therapeutics for use in attenuating the above diseases.